Figure 2: VEGF from BM-MSCs reduces pathology in PNs of NP–C mice.

(a) Protocol of BM-MSC treatment in NP–C mice. (b,c) SphK activity (n=7 per group; b) and VEGF levels (n=8 per group; c) were estimated in the cerebellums of WT and NP–C mice after BM-MSC treatment. (d) Cerebellar sections were stained with anti-calbindin and anti-VEGF (low-magnification scale bar, 50 μm; high-magnification scale bar, 20 μm). Values represent normalized VEGF fluorescence intensities in PCL (n=7 per group). (e) SphK activities were measured in the cerebellums of NP–C mice treated with PBS (n=6), BM-MSCs, VEGF siRNA BM-MSCs and VEGF^tg BM-MSCs (n=8 per group). (f) Left, isolation of mouse PNs using LCM (scale bar, 75 μm). Right, mRNA level of Vegf, VEGFR2 and Sphk1 on LCM-captured PNs samples (n=7 per group). (g) NP–C mice were treated daily with the PTK787 at 100 mg kg⁻¹ or PBS, starting 2 days before the BM-MSC transplantation. One day after BM-MSC treatment, SphK activity was estimated (NP–C, n=7; NP–C BM-MSC TP, n=8 per group). (h) Cerebellar sections were stained with anti-calbindin (scale bar, 50 μm), and the number of calbindin-positive PNs were quantified (n=7 per group). (i) Rota-rod scores of mice were averaged and plotted beginning 3 days after transplantation (n=15 per group). (j) Survival curve of NP–C mice (n=15 per group). Treatment with BM-MSCs and VEGF^tg BM-MSCs resulted in significantly increased survival compared with PBS treatment (P=0.0194 and P=0.0055, respectively; log-rank test). (k) Effect of VEGF knockdown on SphK activity. Left, after intracerebellar injection of control (n=7) or VEGF shRNA (n=8) in mice, SphK activities were measured in the cerebellums. Right, relative levels of Sphk1 mRNA from LCM-captured PNs samples (n=7 per group). b–i, one-way analysis of variance, Tukey’s post hoc test. k, Student’s t-test. *P<0.05, **P<0.01, ***P<0.005. All error bars indicate s.e.m.